Effects of polyurethane matrices on fungal tannase and gallic acid production under solid state culture

Effects of polyurethane matrices on fungal tannase and gallic acid production under solid state culture first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:cag13761@mail.uadec.mx †E-mail:cag13761@mail.uadec.mx cag13761@mail.uadec.mx Department of Food Research, Autonomous University of Coahuila, Saltillo, Coahuila 25000, Mexico Department of Food Research, Autonomous University of Coahuila, Saltillo, Coahuila 25000, Mexico The influence of the physical structure of polyurethane matrix as a support in a solid state culture in tannase production and gallic acid accumulation by Aspergillus niger Aa-20 was evaluated. Three different polyurethane matrices were used as the support: continuous, semi-discontinuous and discontinuous. The highest tannase production at 2479.59 U/L during the first 12 h of culture was obtained using the discontinuous matrix. The gallic acid was accumulated at 7.64 g/L at the discontinuous matrix. The results show that the discontinuous matrix of polyurethane is better for tannase production and gallic acid accumulation in a solid state culture bioprocess than the continuous and semi-discontinuous matrices. Tannase Gallic acid Polyurethane matrix support Solid state culture Aspergillus niger Aa-20 INTRODUCTION 1 Gallic acid (3,4,5-trihydroxy benzoic acid) is a phenolic compound, mainly used in the pharmaceutical industry for manufacturing trimethoprim, an antibacterial agent (Kar and Banerjee, 2000 2000 ). Gallic acid is also used in food industries as a substrate to produce a potent antioxidant, propilgallate. Tannase or tannin-acyl-hydrolase (EC 3.1.1.20), is an extracellular and inducible enzyme, which catalyses the hydrolysis of some tannins and gallic acid esters. Tannins are defined as naturally occurring water-soluble polyphenols of varying molecular weight, which differ from most other natural phenolic compounds in their ability to precipitate proteins from solutions (Lekha and Lonsane, 1997 1997 ). Based on the structures and properties, they are distributed in four groups: condensed tannins, gallotannins, ellagitannins and complex tannins (Khanbabaee and van Ree, 2001 2001 ). Condensed tannins, also known as polymeric proanthocyanidins, are composed of flavonoid units (Bhat et al., 1998 1998 ). Gallotannins are esters of gallic acid linked to a sugar core, usually glucose, whereas ellagitannins are esters of ellagic acid also linked to a sugar core (Aguilar et al., 2007 2007 ). Complex tannins are mixed compounds of flavonoid units linked to gallic acids by ester bonds. Microorganisms are the main source for industrial enzymes due to their biochemical diversities, technical and financial advances. Tannase is usually obtained from bacteria, some yeasts (Aoki et al., 1976 1976 ) and filamentous fungi, mainly from the following species: Aspergillus , Penicillium , Fusarium and Thichoderma (Aguilar et al., 2007 2007 ). This enzyme is applied commercially in the industries of drinks and foods, chemical-pharmaceutics, brewing, and animal feed (Belmares et al., 2004 2004 ). The use of tannase in large scale, however, is much limited due to a variety of factors including production cost and insufficient knowledge of the enzyme (Aguilar and Gutiérrez-Sánchez, 2001 2001 ). Although the gallic acid production in a solid state culture has been evaluated (Kar et al., 1999 1999 ; Kar and Banerjee, 2000 2000 ; Aguilar et al., 2002 2002 ), the influence of the physical nature of a matrix used in a solid culture to produce tannase and gallic acid remains unclear. In this study, we investigate the effects of polyurethane matrices in a solid state culture on Aspergillus niger -induced tannase and gallic acid production. MATERIALS AND METHODS 1 Microorganism 2 Aspergillus niger Aa-20 spores [IRD-UAMI (Research Institute of Development, France and Metropolitan Autonomous University, Mexico) collection] used in this work were stored at −20 °C in cryoblocks® (bead storage system, Technical Service Consultants Ltd., France). Inoculum was prepared by transferring the spores to potato dextrose agar (PDA) and incubated at 30 °C for 3~5 d. The spores were then scraped into a 0.01% Tween 80 solution and counted in a Neubauer chamber. Chemicals 2 Tannic and gallic acids used were analytical grade (Sigma, Mexico, Federal District). PDA agar and salts for Czapek-Dox were obtained from (Merk Reagents, Monterrey, Mexico). Culture medium 2 The medium was Czapek-Dox modified with 12.5 g/L tannic acid as an inducer and a sole carbon source (Aguilar et al., 2002 2002 ). Support 2 Polyurethane foam (PUF) was used as the solid support for the solid culture. PUF matrix was structured in three patterns: continuous, semi-discontinuous and discontinuous. Continuous matrix was constructed by PUF circles of 9.0 cm in diameter and 0.5 cm in thickness; semi-discontinuous matrix was composed of PUF cubes of 0.5 cm×0.5 cm×0.5 cm; and the discontinuous matrix was pulverized PUF. Three grams of each support was placed into a 250 ml Erlenmeyer flask and 7 ml of Czapek-Dox modified media were added. Spores were inoculated at 2×10 7 per gram of PUF, and then were incubated for 48 h at 30 °C, 70% initial humidity, 5.5 initial pH. Six samples were taken during the kinetics. Tannase and gallic acid extracts were recovered by adding 14 ml of water to each reactor. It was filtrated using 41 Whatman paper, and the filtrated liquid was used for the assay of the enzymatic activity. In addition, gallic acid accumulation and substratum consumption were determined as described in the next paragraph. Analytical methods 2 Tannase production was measured by methanolic rhodanine spectrophotometric method (Sharma et al., 2000 2000 ), using methyl gallate prepared in 0.05 mol/L citrate buffer (pH=5) as substrate. The 0.05 mol/L citrate buffer (pH=5) was used as control and also as the crude enzymatic extract. For the colour reaction, the methanolic rhodanine (0.67%) and potassium hydroxide (0.5 mol/L) were used. This reaction was monitored by measuring absorbance at 520 nm. One unit of enzyme (U) was defined as the amount of enzyme able to release 1 mol gallic acid per millilitre per minute. Gallic acid accumulation was also measured by methanolic rhodanine spectrophotometric method (Sharma et al., 2000 2000 ). Tannic acid concentration was evaluated by spectrophotometry using the phenol-sulfuric method ( λ =480 nm) reported by Aguilar ( 2000 2000 ). Briefly the method implies a thermal reaction of 1 ml of sample with 2 ml of phenol-sulfuric reagent (1 mg/ml) during 5 min in a boiling water bath, then the sample is cooled and the absorbance is recorded at 480 nm. Biomass determination was analysed by measuring protein concentration with the biuret assay kit (Randox Laboratories Ltd., USA) following the technique reported by Córdova-López et al.( 1996 1996 ), where 0 g of fermented solid is impregnated with phosphoric acid (0.15 mol/L) and heated in a boiling water bath to hydrolyse the mycelium during 7 min; the sample is then cooled and centrifuged to obtain a mycelial protein solution; finally, 200 ml of sample is mixed with 800 ml of biuret reagent and the blue color is measured at 595 nm. Kinetic parameters 2 Growth curves were fitted by a Maquardt “Solver” computer program (Excel, Microsoft) using logistic equation as follows: $$X = \frac{{X_{\max } }}{{1 + \left( {\frac{{X_{\max } - X_0 }}{{X_0 }}} \right)}}e^{ - \mu t} ,$$ where X (g/L) represents the biomass calculated, X 0 and X max (g/L) are the initial and maximum biomass values, respectively, μ (h −1 ) is the specific growth rate, and t (h) is the culture time. The algorithm minimizes the sum of least square errors, comparing experimental data with the theoretical values obtained. The biomass/substrate yield, Y X / S , is calculated by the next equation: Y X / S =( X max − X 0 )/( S 0 − S final ), where X max and X 0 (g/L) are the maximum and initial biomass values obtained, respectively, and S 0 and S final (g/L) are the initial and final substrate concentration values, respectively. The specific substrate uptake rate, q S , is defined as follows: q S = μ / Y X / S , where q S is giving as grams of substrate consumed per gram of biomass per hour. Tannase/biomass yield, Y E / X , is estimated from the linear correlation between tannase activities, E (U/L) and biomass concentration, X (g/L). The yield coefficient is defined as Y E / X (units of tannase per gram X ). The specific rate of products, q P , is defined as follows: q P = μ / Y P / X , where q P is the units of tannase or grams of gallic acid produced per gram of biomass per hour, Y P / X is the product/biomass yield. RESULTS AND DISCUSSION 1 The biomass production of Aspergillus niger Aa-20 reached the highest value (1.30 g/L) in the semi-discontinuous matrix, whereas discontinuous and continuous matrices gained 0.81 and 0.68 g/L, respectively (Fig. 1 1 ). Table 1 1 presents the values of the kinetic parameters associated with the fungal growth, the specific growth rate µ and the maximum growth X max . µ : Specific growth rate; X max : Maximum biomass values Parameters µ (h −1 ) X max (g/L) Continuous 0.10 0.81 Semi-discontinuous 0.29 1.14 Discontinuous 0.63 0.68 Table 2 2 shows the results of the substrate consumption. It was found that in the continuous matrix the tannic acid was consumed near completely, with only 0.30 g/L remained in the broth at the end of the culture. In the semi-discontinuous matrix, a concentration of 0.70 g/L was remained. However, in the discontinuous matrix, the substrate was not so efficiently consumed, since the remained was at 5.59 g/L. Y X / S : Biomass/substrate yield; q S : Substrate uptake rate (grams of substrate per gram of biomass per hour) Parameters Y X / S q S [g/(g·h)] Continuous 0.07 0.66 Semi-discontinuous 0.11 0.39 Discontinuous 0.10 0.16 Fig. 2 2 presents the results of extracellular tannase production, and Table 3 3 shows the corresponding kinetic parameters. The highest enzyme activity was found at the discontinuous matrix with a 2479.59 U/L value. At the continuous and semi-discontinuous matrices, maximal activities were 543.40 and 479.39 U/L, respectively. The maximal values were obtained at a really short fermentation period around the first 12 h. The culture conditions were: incubation temperature 30 °C, humidity 70%, pH 5.5 and culture time 11.5 h. C: Continuous; SD: Semi-discontinuous; D: Discontinuous Y P / X : Produc/biomass yield; q P : Product formation rate Parameters Y P / X (U/g) q P [U/(g·h)] Continuous 670.86 67.09 Semi-discontinuous 359.46 104.24 Discontinuous 3646.47 2297.28 Lekha and Lonsane ( 1994 1994 ) reported that Aspergillus niger PKL-104 produced 140 000 U/L of tannase in the solid state culture. Kar and Banerjee ( 2000 2000 ) found high amounts of tannase produced by Rhizopus oryzae using a modified-solid fermentation. Also Aguilar et al.( 2001a 2001a ; 2001b 2001b ) reported an 18 000 U/L of tannase as a maximum production. In these three studies, the final productions were affected by many factors including the choices of the enzyme producer microorganism and the solid support, and some culture conditions such as pH, humidity percentage, airing, etc. Fig. 3 3 shows that the discontinuous matrix of PUF favoured the gallic acid accumulation; 7.64 g/L was obtained during the first 12 h of the culture, whereas the continuous and semi-discontinuous matrices produced 0.74 and 0.86 g/L of gallic acid, respectively. The gallic acid accumulation was proportional to the tannase biosynthesis in the production systems (Table 4 4 ). In the discontinuous matrix supported culture system, a high accumulation of gallic acid occurred during the first 10 h, which was associated with the maximum enzymatic activity. The solid state fermentation yielded higher amounts of gallic acid during first hours of the culture, compared with a liquid fermentation (Kar and Banerjee, 2000 2000 ; García-Nájera et al., 2002 2002 ). In this study the gallic acid accumulation at 10 h is 1.2 times higher than the liquid fermentation (García-Nájera et al., 2002 2002 ). Kar and Banerjee ( 2000 2000 ) also reported high concentrations of gallic acid, which, however, were produced on a longer liquid fermentation. The culture conditions were: incubation temperature 30 °C, humidity 70%, pH 5.5 Y P / X : Product/biomass yield; q P : Product formation rate Parameters Y P / X (U/g) q P [U/(g·h)] Accumulation (%) Continuous 0.91 0.09 5.90 Semi-discontinuous 0.66 0.19 6.85 Discontinuous 11.23 7.08 61.13 The use of small particles of PUF favours the formation of fungal aggregates which exhibit a major catalytic activity, while the use of bigger particles of PUF favours the growth of longer hyphaes with a lower catalytic activity. This can be supported by the results obtained by Oostra et al.( 2001 2001 ) and Rahardjo et al.( 2002 2002 ), who demonstrated that the form of fungal cells forming the mycelium in solid state culture is extremely important because it affects the respiration and, therefore, the modulation of enzymes biosynthesis. Also, Viniegra-González and Favela-Torres ( 2006 2006 ) indicated that the diffusion of substrates on a solid support is an important physical parameter that affects the expression of enzymes and it is seriously affected by the continuity of the solid support. In this aspect, Cerda-Montalvo et al.( 2005 2005 ) demonstrated the formation of substrate gradients inside the solid support using PUF in the solid state culture for tannase production. This phenomenon was originally proposed by Georgiou and Shuler ( 1986 1986 ) and confirmed by Mitchell et al.( 1991 1991 ; 2004 2004 ) and Olsson ( 1994 1994 ). Strong glucose gradients were observed by Nagel et al.( 2002 2002 ) in solid state culture and several models have been proposed to explain these profiles of substrates in solid state culture systems by Rajagopalan and Modak ( 1995a 1995a ; 1995b 1995b ), Mitchell et al.( 2004 2004 ), Viniegra-González et al.( 2003 2003 ) and Viniegra-González and Favela-Torres ( 2006 2006 ). The results of the present study show that the discontinuous matrix of polyurethane is better for tannase production and gallic acid accumulation in the solid state culture bioprocess. We conclude that the physical structure of a solid support matrix has a great influence on the physiological behavior of the fungus in the solid culture than the continuous and semi-discontinuous matrices. References CN Aguilar . Induction and Repression of Synthesis of Tannase from Aspergillus niger Aa/20 in Submerged and Solid State Cultures , 2000 . : Mexico Metropolitan Autonomous University , PhD Thesis, (in Spanish) . 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Biochem Eng J , 2003 . 13 ( 2-3 ): 157 - 167 . DOI: 10.1016/S1369-703X(02)00128-6 http://doi.org/10.1016/S1369-703X(02)00128-6 . Advantages of fungal enzyme production in solid state over liquid fermentation systems 10.1631/jzus.2007.B0771 Project supported by the Agricultural, Livestock and Fishing Department (SAGARPA, No. 12385) and the National Forest Commission (CONAFOR, No. 2004-C01-13), and the Council of Science and Technology, Mexico 2007-07-23 2007-08-17 2022-01-05 Copyright © 2007, Journal of Zhejiang University Science 2007 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 08